A MICROINJECTION TECHNIQUE USING DROSOPHILA-MELANOGASTER FOR BIOASSAY-GUIDED ISOLATION OF NEUROTOXINS IN ARTHROPOD VENOMS

Modern analytical techniques permit isolation and structural determina tion of neurotoxins at the picomole level. However, bioassay-guided fr actionation of the sample often relies on simple injection assays usin g insects, vertebrates or crustaceans of a fairly large size, thus con suming quite a large amount of the samples being investigated. In orde r to investigate samples of very small size, we have devised an insect microinjection method using glass micropipettes and Drosophila melano gaster adults as test insects. The validity of the method was tested w ith a series of six buthoid scorpion venoms (Androctonus australis, Bu thotus judaicus, Buthus tamulus, Centruroides sculpturatus, Leiurus qu inquestriatus hebraeus, Tityus serrulatus) and one chactoid scorpion ( Scorpio maurus palmatus) as standards. The LD(50)S Of the venoms were determined using both the microinjection method and a classical inject ion assay with crickets (Gryllus bimaculatus) as test insects. Results demonstrated that the new method can successfully be applied to the s tudy of insect neurotoxic activity in arthropod venoms. The Gryllus:Dr osophila ratio in amount of sample utilized is 100. However, for all B uthoid venoms tested, except L. quinquestriatus, Drosophila showed les s sensitivity, thus reducing the gain by a factor of 2-10. Drosophila were several times more sensitive to the only chactoid venom tested. T hese results clearly demonstrate the advantage of using this microtech nique, when limited amounts of material are available for both chemica l and biological work.